Genetic polymorphisms in genes regulating cell death and prognosis of patients with rectal cancer receiving postoperative chemoradiotherapy

Objective: The identification of biomarkers for predicting chemoradiotherapy efficacy is essential to optimize personalized treatment. This study determined the effects of genetic variations in genes involved in apoptosis, pyroptosis, and ferroptosis on the prognosis of patients with locally advanced rectal cancer receiving postoperative chemoradiotherapy (CRT). Methods: The Sequenom MassARRAY was used to detect 217 genetic variations in 40 genes from 300 patients with rectal cancer who received postoperative CRT. The associations between genetic variations and overall survival (OS) were evaluated using hazard ratios (HRs) and 95% confidence intervals (CIs) computed using a Cox proportional regression model. Functional experiments were performed to determine the functions of the arachidonate 5-lipoxygenase (ALOX5) gene and the ALOX5 rs702365 variant. Results: We detected 16 genetic polymorphisms in CASP3, CASP7, TRAILR2, GSDME, CASP4, HO-1, ALOX5, GPX4, and NRF2 that were significantly associated with OS in the additive model (P < 0.05). There was a substantial cumulative effect of three genetic polymorphisms (CASP4 rs571407, ALOX5 rs2242332, and HO-1 rs17883419) on OS. Genetic variations in the CASP4 and ALOX5 gene haplotypes were associated with a higher OS. We demonstrated, for the first time, that rs702365 [G] > [C] represses ALOX5 transcription and corollary experiments suggested that ALOX5 may promote colon cancer cell growth by mediating an inflammatory response. Conclusions: Polymorphisms in genes regulating cell death may play essential roles in the prognosis of patients with rectal cancer who are treated with postoperative CRT and may serve as potential genetic biomarkers for individualized treatment.


Introduction
Colorectal cancer (CRC), a major malignant disease of the gastrointestinal tract, has the third highest incidence among cancers and is the second leading cause of cancer deaths 1 . The incidence of CRC ranks fourth among men and third among women in China 2 . CRC is caused by interactions between genetic and environmental factors 3 . Pathogenic and putative pathogenic germline mutations can increase the risk of CRC 4 . Rectal cancer accounts for approximately 30% of CRC cases; however, the treatment for resectable stage II and III rectal cancer is different from colon cancer, largely because of the different local recurrence patterns 5,6 . Postoperative chemoradiotherapy (CRT) is often administered to patients with locally advanced rectal cancer; however, the efficacy varies from one individual to another 7 . Therefore, the identification of prognostic markers is important for personalized treatments.
Cell death can be categorized into accidental death (ACD) and regulated death (RCD) 8 . ACD is a biological process in which cells die uncontrollably, whereas RCD is regulated by a set of molecules. Under physiologic conditions, RCD is also known as programmed cell death (PCD). RCD includes apoptosis, pyroptosis, and ferroptosis, which have been widely studied and exhibit unique molecular mechanisms 9 . Apoptosis is a well-recognized form of PCD that is essential for the normal development and function of organisms; however, aberrant apoptosis is associated with many diseases, including cancer and autoimmune diseases 10 . Radiotherapy and chemotherapy induce tumor cell death mainly through caspase-dependent apoptosis 11,12 ; however, radiotherapy and chemotherapy also induce pyroptosis and ferroptosis to exert anti-tumor effects [13][14][15][16][17][18] . Pyroptosis is an inflammatory RCD in which plasma membrane pores are formed by members of the gasdemin protein family that are often cleaved by activated caspases, such as caspase-1 (CASP1), caspase-4 (CASP4), caspase-5 (CASP5), and caspase-11 (CASP11) 9,19 . Ferroptosis is a form of RCD caused by phospholipid peroxidation, which is dependent on iron, reactive oxygen species (ROS), and phospholipids containing polyunsaturated fatty acid chains (PUFA-PL) 20,21 . Ferroptosis is regulated by the antioxidant enzyme glutathione peroxidase 4 (GPX4), iron chelators, and antioxidants 22,23 . Induction of ferroptosis holds great promise in tumor therapy because the inducers of ferroptosis were discovered in the process of searching for new cancer therapeutic compounds 24,25 .
Single-nucleotide polymorphisms (SNPs) are associated with the prognosis of patients with cancer. We have previously shown that genetic variations, such as SNPs in microRNA and DNA repair genes, are significantly associated with the prognosis of patients with locally advanced rectal cancer who are treated with postoperative CRT 26,27 . Moreover, polymorphisms in genes involved in the apoptosis and ferroptosis pathways are associated with the overall survival (OS) of patients with cancer [28][29][30] ; however, previous studies only focused on one or two genes. A comprehensive understanding of the association between OS and ferroptosis and apoptosis pathways is limited.
In the present study we investigated the associations between haplotype-tagging SNPs (htSNPs) of key genes involved in the apoptosis, pyroptosis, and ferroptosis pathways with the OS of rectal cancer patients treated with postoperative CRT. We analyzed the associations with single locus, combined multiple loci, and haplotypes in these three pathways. What's more, this is the first study to demonstrate that arachidonate lipoxygenase 5 (ALOX5) rs702365 [G] > [C] represses ALOX5 transcription and leads to a decrease in ALOX5 expression.

Patient characteristics and data collection
Three hundred patients with rectal cancer receiving concurrent CRT were enrolled in this study. The patient characteristics have been described in our previous studies 26,27 . All patients signed an informed consent form. This study was approved by the Institutional Review Board of the Cancer Hospital (Chinese Academy of Medical Sciences; IRB No. NCC2019C-145) and met the Declaration of Helsinki requirements. Briefly, the criteria for enrollment were as follows: (1) diagnosis of rectal adenocarcinoma by pathology experts at the Cancer Hospital of the Chinese Academy of Medical Sciences from January 2005 to June 2015; (2) primary and locally advanced rectal cancer without distant metastasis; (3) Karnofsky performance score (KPS) ≥ 70 and life expectancy ≥ 6 months; (4) age ≤ 75 years; (5) normal routine blood and biochemistry tests before concurrent CRT; (6) no history of CRT or other tumors; and (7) the patient underwent total mesorectal excision surgery and concurrent CRT. The total radiation dose was as follows: 50 Gy; 2 Gy/session; and 5 sessions/week for 5 weeks. The chemotherapeutic regimen was capecitabine (1,600 mg/m 2 daily administered orally twice for 2 weeks, stopped for 1 week, and continued for 2 weeks).
The relevant clinical data of the patients were obtained from medical records, hematologic tests, imaging results, and telephone inquiries about cancer-related health status after discharge from the hospital. The last follow-up date was April 1, 2021.
The genetic variation screening strategy is shown in Supplementary Figure S1. Among the loci of 40 candidate genes, SNPs with a minor allele frequency (MAF) ≥ 0.1 in the Chinese Han Beijing population (CHB) of the Thousand Genomes Project (1,000 Genomes) query were first selected. For SNPs with a correlation coefficient (r 2 ) ≥ 0.8, only 1 SNP was selected as the htSNP based on linkage disequilibrium (LD) information of the CHB in the database, and 217 genetic variations were selected for genotyping. Finally, we analyzed the associations of 169 SNPs with the following criteria: Hardy-Weinberg equilibrium (HWE) > 0.05; MAF ≥ 0.10; and call rate ≥ 95%.

Genotyping
Genomic DNA was extracted from blood samples (2 mL) collected from each patient before CRT. Genotyping was performed using the Sequenom MassARRAY method, with one blank sample and four duplicate samples arranged in a 96-well plate for quality control.

Cell lines and reagents
HCT8 and HCT116 cells were purchased from the Cell Bank of the Institute of Basic Medical Sciences (Chinese Academy of Medical Sciences) and the School of Basic Medicine (Peking Union Medical College). These cell lines were tested for Mycoplasma contamination and authenticated using STR profiling. HCT8 cells were maintained in RPMI-1640 medium, whereas HCT116 cells were maintained in DMEM with 10% fetal bovine serum in a 5% CO 2 humidified atmosphere at 37°C.

Construction of plasmids, transient transfection, and dual-luciferase reporter assays
A total of 540-bp DNA fragments surrounding the functional candidate SNP rs702365 [G] or [C] alleles were cloned into the pGL4.10-SV40 firefly luciferase expression vector. To determine whether the DNA fragment containing the rs702365 [G] or [C] allele had different ALOX5 promoter-driving abilities, the SV40 sequence in the plasmid was replaced with the ALOX5 promoter (Supplementary Table S1). The authenticity of all constructs was verified by DNA sequencing. The primers used for plasmid construction are listed in Supplementary  Table S1. For dual-luciferase reporter gene assays, 8 × 10 4 HCT8 cells or 1 × 10 5 HCT116 cells were seeded in 48-well plates and transfected with 300 ng of allele-different reporter constructs per well using Lipofectamine 2000 (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) after 16 h. The Renilla luciferase plasmid, pRL-TK (6 ng; Promega, Madison, WI, USA), was co-transfected in each well as an internal control. After a 24 h transfection, the cells were collected and analyzed for luciferase activity. Each plasmid construct had three replicates and was used in at least three independent experiments. The pGL4.10-SV40 vector and pRL-TK plasmid maps are shown in Supplementary Figure S2A, S2B.

Electrophoretic mobility-shift assays (EMSAs)
Nuclear proteins were extracted from HCT8 and HCT116 cells using a Nuclear Protein Extraction Kit (Thermo Fisher Scientific). Additionally, 25-bp double-stranded oligonucleotides containing rs702365 [G] or rs702365[C] were synthesized and labeled with biotin at the 5′ end (Supplementary Table S1). Nuclear extracts (10 μg of protein) were incubated with 100 fmol of biotin-labeled oligonucleotide probes for 20 min using the Chemiluminescent EMSA kit (Beyotime Biotechnology, Shanghai, China). Unlabeled oligonucleotides were added before adding biotin-labeled probes for competition assays. After electrophoreses at 110 V in 0.5 × TBE for 95 min, samples were transferred onto a nylon membrane (Millipore Sigma, Burlington, VT, USA) in 0.5 × TBE at 380 mA for 40 min. The transferred DNA was cross-linked to the membrane at 120 mJ/cm 2 for 90 s, then detected using the ECL reagent in the EMSA kit.

Small-interfering RNA transduction and quantitative real-time polymerase chain reaction (RT-qPCR) assays
HCT8 (3 × 10 5 ) or HCT116 (6 × 10 5 ) cells were seeded in 6-well plates and transfected with small-interfering RNAs (siRNAs) targeting ALOX5 or a negative control (siControl) siRNA using Lipofectamine 2000 according to the manufacturer's protocol. An RNA-Quick Purification Kit (RN001; ES Science, Shanghai, China) was used to extract total RNA from the cells. PrimeScript RT reagent kits and SYBR Premix Ex Taq II kits (Takara Bio, Inc., Shiga, Japan) were used to detect mRNA expression, which was normalized to GAPDH.

Malondialdehyde (MDA) detection
MDA is a lipid peroxidation marker 31 . The MDA level was detected using an MDA assay kit (Dojindo, Beijing, China). In brief, the standard curve was plotted according to the technical manual of the MDA kit. An MDA-TBA adduct is formed by the reaction of MDA in the sample and thiobarbituric acid (TBA), which can be quantified by fluorescence intensity using a microplate reader (Ex: 540 nm, Em: 590 nm). Then, the MDA concentration can be calculated based on the standard curve.

Cell death assay
Cells were cultured in 6-well plates in the presence or absence of 5-fluorouracil (Selleck, Shanghai, China) and irradiation (2 Gy) treatment (CT/RT). The 5-fluorouracil was dissolved in dimethyl sulfoxide (DMSO) (Millipore Sigma). After a 48 h treatment, cells were collected and resuspended, then stained with propidium iodide (PI) (Dojindo) for 15 min, followed by flow cytometric analysis.

Cell viability
Cells transfected with siRNA or siControl were seeded in 96-well plates. The cells were measured daily for 96 h using a Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan). CCK-8 reagent (10 μL) was added to each well and incubated at 37°C for 1.5 h. Absorbance was measured at 450 nm.

Statistical analysis
The HWE of the genotypes was determined using a chi-square (χ 2 ) test. OS was calculated from the date of diagnosis until the time of death or the last follow-up visit. The associations between OS and genetic variations were evaluated using adjusted hazard ratios (HRs) with 95% confidence intervals (CIs), and calculated using Cox regression models and adjusted for gender, age, tumor stage, tumor grade, KPS, surgical procedure, and tumor location as covariates. Kaplan-Meier survival analysis was used to estimate the survival distributions, and the differences between groups were compared using the logrank test. Haplo.stats (version 1.8.7) in the R package was used to estimate haplotype frequencies. Student's t-test was used to determine the difference between the two groups. All statistical analyses were performed using R software (version 4.0.5) and SPSS (version 25). Statistical significance was set at P < 0.05.

Clinical characteristics of patients
The clinical characteristics of the patients are summarized in Table 1. At the time of the final analysis (April 1, 2021) the median follow-up duration was 114 months (range, 10-195 months). Among the 300 patients included in this study, 226 (75.3%) survived and 74 (24.7%) died, 126 (42.0%) had stage II tumors and 174 (58.0%) had stage III tumors, and 196 (65.3%) had tumors ≥ 5 cm from the anus and 104 (34.7%) had tumors < 5 cm from the anus. Dixon surgery was performed in 238 (79.3%) patients, whereas Mile's surgery was performed in 62 (20.7%) patients. Additionally, 12 (4.0%) tumors were poorly graded, whereas 259 (86.3%) and 29 (9.7%) were moderately and well-graded, respectively. Moreover, 17 (5.7%) patients had a score of 70, and 151 (50.3%) and 132 (44.0%) patients had scores of 80 and 90-100, respectively. The median survival time was significantly shorter in patients with stage III rectal cancer than those with stage II cancer (P < 0.001). Moreover, patients with low KPS scores had shorter median survival times than patients with high KPS scores (P = 0.008). There were no

Associations between combined SNPs and OS
We evaluated the additive effects of 3 SNPs (CASP4 rs571407, ALOX5 rs2242332, and HO-1 rs17883419) that had the smallest P-value in the dominant model ( Table 2). We selected the risk genotypes according to the results shown in Table 2, as follows: TT for rs571407; CC for rs2242332; and CT + TT for rs17883419. Compared to patients without the unfavorable genotypes, patients carrying 1, 2, or 3 unfavorable genotypes had an adjusted HR of 3.32 (95% CI = 1.51-7.27; P = 0.003), 4.66 (95% CI = 2.08-10.47; P < 0.001), or 10.62 (95% CI = 4.04-27.93; P < 0.001), respectively (Table 3), with a P value < 0.001 in the trend test, thus suggesting a significant cumulative effect of these SNPs. When patients with 2 or 3 risk genotypes were combined for analysis, the adjusted HR for mortality was 5.57 (95% CI = 2.55-12.16; P < 0.001) compared to patients without risk genotypes. Kaplan-Meier survival curves based on the number of unfavorable genotypes are shown in Figure 1J, 1K.
We have previously shown that hsa-miR-4274 rs202195689, PMS1 rs5743030, rs4920657, and rs5743100 SNPs are significantly associated with OS (Supplementary Table S3 and Supplementary Figure S3H-S3K) 26,27 . In the present study, we jointly analyzed these two SNPs (rs202195689 and rs5743030) with the three above-mentioned SNPs and showed that the HR for mortality increased as the number of risk genotypes increased (P trend < 0.001; Supplementary Table S4). Compared to patients without the risk genotype or carrying 1 risk genotype, the HRs of mortality for patients with 2, 3, or ≥ 4 risk genotypes were 2.58 (95% CI = 1.08-6.13; P = 0.033),  Figure 1L and Supplementary Figure S3L.

ALOX5 may promote proliferation via an inflammatory response in colon cancer cells
It has been reported that activation of ALOX5 leads to lipid peroxidation and oxidative cell death 32,33 . We verified that finding in colon cancer cell lines and found that lipid peroxide and the cell death percentage were reduced in HCT8 and HCT116 cells treated with CT/RT after ALOX5 knockdown by siRNAs (Supplementary Figure S4A-S4D).
To further determine the reason why decreased expression of ALOX5 can improve the prognosis of rectal cancer patients, we continued to transfect ALOX5 siRNAs into HCT8 and HCT116 cells to investigate whether ALOX5 plays a role in CRC tumorigenesis. Additionally, a CCK-8 assay was performed to determine whether ALOX5 promotes the growth of HCT8 and HCT116 cells. Cell proliferation was significantly inhibited in the si-ALOX5 group ( Figure 3A). RT-qPCR was used to confirm the efficiency of si-ALOX5 interference with ALOX5 in HCT8 and HCT116 cells (Figure 3B).
To investigate the possible mechanism underlying the effect of ALOX5, a total of 1,895 genes co-expressed with ALOX5 that were identified by the cBioPortal website using CRC data from the TCGA database, were applied for Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis and Gene Ontology (GO) functional annotation analysis online on the Metascape website. Enrichment analysis revealed that these genes were mainly enriched in inflammatory and immune responses (Figure 3C). Relative luciferase activity C ALOX5 chr10_45438883_C_G_b38 Colon -Sigmoid ALOX5 chr10_45442791_T_C_b38 Colon -Sigmoid P = 6.7e-5 P = 1.9e-8 ALOX5 chr10_45447987_A_T_b38 Colon -Sigmoid P = 1. Inflammation plays a key role in the occurrence and development of CRC 34,35 . Therefore, we determined the expression of the pro-inflammatory mediator, inducible nitric oxide synthase (iNOS), and the inflammatory gene-related transcription factor, NF-κB [36][37][38] . The expression of p-NF-κB p65 was decreased in the ALOX5 knockdown group compared to the control group (Figure 3D). These results suggested that ALOX5 promotes CRC progression by mediating the inflammatory response. In summary, our findings indicated that the ALOX5 rs702365 [G] > [C] change has a tumor suppressive role in CRC by decreasing ALOX5 transcription ( Figure 3E).
In this study we investigated the associations between 169 genetic variations in 40 genes involved in apoptosis, pyroptosis, and ferroptosis with the OS of locally advanced rectal cancer patients receiving postoperative CRT and identified 16 significantly associated genetic variations. Among the genetic variations, 5 (CASP3 rs113420705 and rs4647700, CASP7 rs12263370, GSDME rs2954558, and HO-1 rs17883419) were associated with a shorter OS and 11 (TRAILR2 rs11784599; CASP4 rs571407, rs612987, rs623114, and rs543923; ALOX5 rs702365, rs2242332, and rs4948673; GPX4 rs36207883; HO-1 rs2071749; and NRF2 rs73976300) were associated with a longer OS. Three SNPs (CASP4 rs571407, ALOX5 rs2242332, and HO-1 rs17883419) exhibited cumulative effects, which increased the risk of mortality with an increased number of high-risk genotypes. Additionally, the genetic variations in CASP4 and ALOX5 haplotypes were associated with a higher OS. We also revealed, using functional annotation and prediction through GTEx, RegulomeDB, and GWAVA, that rs702365 was likely a functional SNP. Further biochemical experiments suggested that the rs702365 variant may regulate ALOX5 expression via a long-range regulatory mechanism and influence the proliferation of colon cancer cells.
CASP3 has important roles in apoptosis and pyroptosis. CASP3 is a downstream effector of the caspase cascades in the apoptosis pathway and cleaves gasdermin E (GSDME) to induce pyroptosis 13 . The CASP3 rs1049253 TT genotype is significantly associated with longer second primary malignancy-free survival compared to the TC/CC genotypes 49 .
A CASP3 rs113420705 C > T change decreases the risk of lung cancer 50 . Additionally, CASP4 triggers pyroptosis by cleaving gasdermin D 51 and may act as a tumor suppressor in gastric cancer and esophageal squamous cell carcinoma 52,53 . In contrast, a recent study has shown that high CASP4 expression is associated with poor survival and decreased sensitivity to chemotherapy in clear cell carcinoma 54 . In the present study we demonstrated that 4 CASP4 SNPs (rs571407, rs612987, rs623114, and rs543923) were associated with a longer OS time, and the haplotypes, CCGC (H2) and TCGC (H3), were associated with a reduced risk of mortality compared to the haplotype, CTAT (H1), in patients with rectal cancer receiving postoperative CRT. Therefore, further investigation of these SNPs in CASP4 is required to determine the function in rectal cancer.
The main mechanism underlying the ferroptosis effect is that given the function of iron or lipoxygenases (LOXs), the highly expressed polyunsaturated fatty acids (PUFAs) on the cell membrane are catalyzed to generate lipid ROS, which induce cell death 21,55 . Among the six arachidonate LOXs identified in humans, ALOX5 has an important role in leukotriene (LTs) synthesis 56 . Activation of ALOX5 occurs selectively in ferroptosis-sensitive cells, leading to lipid peroxidation and oxidative cell death 33 . Additionally, ALOX5 activity is critical for the inflammatory response. ALOX5 is required for the recruitment of eosinophils in the abdominal cavity and blocking ALOX5 inhibits inflammatory and immune responses 57 . ALOX5 participates in the biosynthesis of LTs, which are important inflammatory mediators causing inflammatory symptoms that include the accumulation of leukocytes. Several studies have been conducted on the association between ALOX5 and cancer susceptibility or survival [58][59][60][61] . ALOX5 rs2115819 and rs12264801 are associated with poor survival in ovarian cancer 59 . A variable nucleotide tandem repeat polymorphism in ALOX5 promoter is significantly associated with a lower risk of rectal cancer 61 . In addition, there are a few studies on the function of ALOX5 in CRC 62,63 . ALOX5 is upregulated in colon cancer, and its inhibition suppresses CRC progression through the PI3K/AKT pathway 63,64 . In the present study we showed that the rs702365 [C] allele altered the transcriptional activity, which subsequently decreased ALOX5 expression, leading to the inhibition of lipid peroxidation and cell death after CT/RT treatment; however, the rs702365 [C] allele also inhibits the inflammatory response and proliferation of colon cancer cells, thereby having a protective role.
In recent years a few studies have been published on the relationship between genetic variations and the risk of cancer or patient survival. We have previously reported that the rs202195689 SNP in the microRNA seed region and rs5743030, rs4920657, and rs5743100 SNPs in PMS1 are associated with OS in patients with rectal cancer, indicating that these variants may predict the prognosis of patients receiving postoperative CRT 26,27 . Notably, the present study is the first study to comprehensively evaluate the effect of genetic variations in genes related to the three well-known RCD pathways on the prognosis of patients with rectal cancer receiving postoperative CRT.
There were a few limitations to the present study. First, all the subjects involved in this study were recruited from the same hospital, thus selection biases cannot be ignored. Second, the sample size was not large enough.

Conclusions
In summary, we identified 16 genetic variations in genes related to apoptosis, pyroptosis, and ferroptosis that were associated with the prognosis of patients with rectal cancer receiving postoperative CRT. Our results suggest that these SNPs may serve as potential prognostic biomarkers for patients with rectal cancer. We demonstrated that ALOX5 rs702365 [G] > [C] substitution leads to decreased ALOX5 expression. Moreover, preliminary in vitro experiments suggested that reduced ALOX5 expression decreased the proliferative ability of colon cancer cells by inhibiting an inflammatory response, ultimately suppressing the development of colon cancer, which should be thoroughly tested by more functional assays. Nevertheless, our results should be further validated by a large-scale study, and the functional molecular mechanisms of the other SNPs also require further investigation.